System and method

ABSTRACT

A system and method of characterizing, in particular sequencing, polymeric samples, the system comprising: a space-time map generator for generating a space-time map of points representative of the signal peaks of signals detected at a plurality of spaced positions as passed by the migrating components of a plurality of separately-provided sample plugs; and a vertex finder for identifying vertices from the space-time map, with a single vertex being identified for the components of each separately-provided sample plug and the velocities of the sample components being determinable from the points in the space-time map.

[0001] The present invention relates to a system for and method ofcharacterising, in particular sequencing, polymeric samples, inparticular, but not exclusively, as measured electrophoretically. Thetechnique of the present invention is the MITSO™ characterisationtechnique.

[0002] Electrophoretic separation techniques are separation techniquesin which the components of a sample plug are separated in a separationcolumn by the differences in the migration rates of those samplecomponents on the application of an electric field therealong, whereabsorption, fluoroescence, electrochemistry, conductivity, radioactivityand mass spectrometry can be all used to detect the electrophoreticseparation.

[0003] By way of example, DNA samples are commonly characterised bySanger sequencing. Whilst Sanger sequencing is in common use, thetechnique is limited in a number of respects, not least because the DNAbands have to be labelled according to base pair termination.

[0004] It is an aim of the present invention to provide an improvedsystem for and method of characterising in particular sequencing,polymeric samples, such as DNA samples, which can be used with bothlabelled and non-labelled sample components.

[0005] Accordingly, the present invention provides a system forcharacterising, in particular sequencing, polymeric samples, comprising:a space-time map generator for generating a space-time map of pointsrepresentative of the signal peaks of signals detected at a plurality ofspaced positions as passed by the migrating components of a plurality ofseparately-provided sample plugs; and a vertex finder for identifyingvertices from the space-time map, with a single vertex being identifiedfor the components of each separately-provided sample plug and thevelocities of the sample components being determinable from the pointsin the space-time map.

[0006] Preferably, the system further comprises a velocity sorter fordetermining the nominal velocities associated with the signal peaks inthe signals and grouping those signal peaks into sets according tonominal velocity.

[0007] Preferably, the space-time map generator is configured to utilisea corrected time component in generating the space-time map according toa function of the electric current variation.

[0008] More preferably, the correction is according to the functiont_(c)=∫I₀/I(t′)dt′ in the range of 0 to t, where t is the measured time,t_(c) is the corrected time, I is the measured current and I₀ is thereference current.

[0009] Preferably, the space-time map generator is an equiphasespace-time map generator for generating an equiphase space-time map ofequiphase points.

[0010] More preferably, the equiphase space-time map generator isconfigured to transform each data set into a set of local slopes anddetermine the local minima as the minimum absolute local derivatives.

[0011] In one embodiment the components are non-labelled.

[0012] In another embodiment the components are labelled.

[0013] Preferably, the components are migrated through a channel.

[0014] More preferably, the channel comprises a separation channelthrough which the components are electrophoretically driven.

[0015] Preferably, the sample plugs respectively comprise DNA bandshaving one of the base pair terminations.

[0016] The present invention also extends to an electrophoresisapparatus including the above-described system.

[0017] The present invention also provides a method of characterising,in particular sequencing, polymeric samples, comprising the steps of:generating a space-time map of points representative of the signal peaksof signals detected at a plurality of spaced positions as passed by themigrating components of a plurality of separately-provided sample plugs;and identifying vertices from the space-time map, with a single vertexbeing identified for the components of each separately-provided sampleplug and the velocities of the sample components being determinable fromthe points in the space-time map.

[0018] Preferably, the method further comprises the steps of determiningthe nominal velocities associated with the signal peaks in the signalsand grouping those signal peaks into sets according to nominal velocity.

[0019] Preferably, a time component corrected according to a function ofthe electric current variation is utilised in generating the space-timemap.

[0020] More preferably, the correction is according to the functiont_(c)=∫I₀/I(t′)dt′ in the range of 0 to t, where t is the measured time,t_(c) is the corrected time, I is the measured current and I₀ is thereference current.

[0021] Preferably, the space-time map is an equiphase space-time map ofequiphase points.

[0022] More preferably, the equiphase points are determined bytransforming each data set into a set of local slopes and determiningthe local minima as the minimum absolute local derivatives.

[0023] In one embodiment the components are non-labelled.

[0024] In another embodiment the components are labelled.

[0025] Preferably, the components are migrated through a channel.

[0026] More preferably, the channel comprises a separation channelthrough which the components are electrophoretically driven.

[0027] In one embodiment the sample plugs are provided at spaced timeintervals.

[0028] In another embodiment the sample plugs are provided at spacedpositions.

[0029] Preferably, the sample plugs are provided at substantially thesame time.

[0030] Preferably, the sample plugs respectively comprise DNA bandshaving one of the base pair terminations.

[0031] A preferred embodiment of the present invention will now bedescribed hereinbelow by way of example only with reference to theaccompanying drawings, in which:

[0032]FIG. 1 illustrates the detector chip of an electrophoresisapparatus in accordance with a preferred embodiment of the presentinvention;

[0033]FIG. 2 illustrates the analysis system of the apparatus of FIG. 1;

[0034]FIG. 3 illustrates a three-dimensional representation of theintensity-time signals of one component of a sample plug as detected atpositions z₁, z₂, z₃ spaced along the separation channel of theapparatus of FIG. 1;

[0035]FIG. 4 illustrates the intensity-time signals of three componentsof a sample plug as detected at positions z₁, z₂, z₃ spaced along theseparation channel of the apparatus of FIG. 1;

[0036]FIG. 5 illustrates a space-time map as generated from theintensity-time signals of FIG. 4;

[0037]FIG. 6 illustrates the velocity spectrum as determined from thevertexed space-time map of FIG. 5; and

[0038]FIG. 7 illustrates a space-time map as generated from theintensity-time signals from four separately-injected DNA sample plugscomprising DNA bands having different base pair terminations.

[0039]FIGS. 1 and 2 illustrate an electrophoresis apparatus inaccordance with a preferred embodiment of the present invention.

[0040] The electrophoresis apparatus includes a detector chip 2 asmicrofabricated in a substrate chip, and an analysis system 3 foranalysing the detection signals generated by the detector chip 2.

[0041] The detector chip 2 includes a separation channel 4, in thisembodiment a meandering, gel-filled channel, through which thecomponents of one or more sample plugs are in use driven by an appliedelectrophoretic voltage. The separation channel 4 has a lengthsufficient to allow separation of the components of the sample plugs.Preferably, the separation channel 4 has a width of from 25 to 100 μmand a length of from 20 to 300 mm. The separation channel 4 includes aplurality, in this embodiment first to fourth, spaced sample-injectionports 6, 8, 10, 12 through which sample plugs including a plurality ofcomponents, in this embodiment DNA bands having the respective base pairterminations A, T, G and C, are separately injected into the separationchannel 4.

[0042] The detector chip 2 further includes a light source 14, in thisembodiment a UV light source, disposed along a length of one side of theseparation channel 4, and a detector 16 disposed along the length of theother side of the separation channel 4 to detect light transmittedthrough the separation channel 4, with the presence of the migratingcomponents being detected by the change in the detected light intensityas caused by absorbtion of the incident light. By detecting the samplecomponents in this manner, the sample components need not necessarily belabelled. In this embodiment the detector 16 comprises a pixel detectorarray (PDA) which includes a plurality of pixels providing detectingelements for detecting the transmitted light at a plurality of positionsz₁, z₂, z₃ spaced along the length of the separation channel 4 andoutputting a plurality of signals S₁, S₂, S₃. For ease of description,the detector 16 is illustrated as including three detecting elements atthree positions z₁, Z₂, Z₃. It will, however, be understood that inpractice the detector 16 comprises a plurality of detecting elements ata plurality of positions z₁, z₂, z₃, . . . ,Z_(n), which each output asignal S₁, S₂, S₃, . . . , S_(n). In an alternative embodiment thedetector 16 could be provided by a plurality of separate detectors eachproviding a detecting element. In another alternative embodimentlabelled sample components could be used, such as sample componentsincluding fluorescent or radioactive labels, which labels would bedetected by the detector 16.

[0043] The analysis system 3 comprises a data collector 18 for receivingthe signals S₁, S₂, S₃ generated by the detector 16 and storing thosesignals S₁, S₂, S₃ as data sets, a velocity sorter 19 for determiningthe nominal velocities v₁, v₂, v₃ of the sample components associatedwith each of the signal peaks SP₁, SP₂, SP₃ of each of the signals S₁,S₂, S₃ and grouping those signal peaks SP₁, SP₂, SP₃ into sets accordingto nominal velocity, an equiphase space-time map generator 20 forgenerating an equiphase space-time map of equiphase points from thesignal peaks SP₁, SP₂, SP₃ of the signals S₁, S₂, S₃, and a vertexfinder 22 for identifying the vertices of the equiphase points of thegrouped sets of signal peaks SP₁, SP₂, SP₃. In this embodiment thevelocity sorter 19 is provided so as to be operable prior to theequiphase space-time map generator 20. In alternative embodiments thevelocity sorter 19 could be provided so as to be operable after thespace-time map generator 20 or the vertex finder 22.

[0044]FIG. 3 is included for the purposes of illustration only andillustrates the signals S₁, S₂, S₃ as including only a single peak SP₁,from a single component of a single sample plug. In reality, however.the signals S₁, S₂, S₃ each include a plurality of signal peaksSP_(1-n), SP_(1-n), SP_(1-n). FIG. 4 illustrates the signals S₁, S₂, S₃as including three signal peaks SP₁, S₂, SP₃ from three components of asingle sample plug.

[0045] The velocity sorter 19 is configured to determine the nominalvelocities v₁, v₂, v₃ of the sample components associated with each ofthe signal peaks SP₁, SP₂, SP₃ in each of the signals S₁, S₂, S₃ andthen group those signal peaks SP₁, SP₂, SP₃ into sets according tonominal velocity. The nominal velocities v₁, v₂, v₃ can be calculated asthe positions z₁, Z₂, Z₃ of the detector elements are fixed and theelapsed time t is extractable from the signals S₁, S₂, S₃, where thenominal velocities can be expressed as v_(1-n)=z_(1-n)/t. By groupingthe signal peaks SP₁, SP₂, SP₃ into sets according to nominal velocity,and hence sample component, subsequent analysis is facilitated as thedata points associated with each sample component can be fitted withoutrequiring the use of complex data extraction techniques. Velocitysorting is encompassed by our earlier WO-96/35946, the content of whichis incorporated herein by reference.

[0046] The equiphase space-time map generator 20 is configured todetermine the local minima of the signal peaks SP₁, SP₂, SP₃ in thesignals S₁, S₂, S₃ detected at the detection positions z₁, Z₂, Z₃ andgenerate an equiphase map M in space-time dimensions from the determinedlocal minima. FIG. 5 illustrates the space-time map M generated from thelocal minima extracted from the signal peaks SP₁, SP₂, SP₃ of thesignals S₁, S₂, S₃.

[0047] In this embodiment each electropherogram is transformed into aset of local slopes, where a triangular slope sequence defines a signaland the local extreme is the minimum absolute local derivative.

[0048] Also, in this embodiment the time component of the detectedsignals S₁, S₂, S₃ is corrected as a function of the integrated electriccurrent variation. Owing to the variation of various factors inelectrophoretic detection, the temperature being one of the mostsignificant, the characteristics of the separation medium, in thisembodiment a gel, are altered. Firstly, the resistivity of the gelchanges, leading to variations in the potential difference between theelectrodes and a given point in the gel and fluctuations in the electriccurrent. Secondly, the sieving properties of the gel change, affectingthe mobility of the electrophoresed components. By monitoring theelectric current, the time component of the space-time map M can becorrected as set out hereinbelow. Specifically, the time component iscurved as a function of the integrated electric current variation.

[0049] The velocity of a sample component is:

v=dz/dt  (1)

[0050] For a transformation of the measured time component to acorrected time component t→t_(c), it follows that dt→dt_(c) and v→v_(c).Thus:

v _(c) /v=dt/dt _(c)  (2)

[0051] The transformation v→v_(c) can be defined as:

v _(c) /v=I(t)/I ₀  (3)

[0052] where I is the measured current and I₀ is the reference currentwhich corresponds to the frame where all velocities and time componentsare projected.

[0053] From equations (2) and (3), it follows:

dt _(c) =I ₀ /I(t)dt→t _(c) =∫I ₀ /I(t′)dt′ for 0 to t  (4)

[0054] The justification for the velocity transformation (3) is that thevelocity is approximately proportional to the applied electric field,which in turn is proportional to the electric current in the separationchannel 4. This correction factor has been found to work well for smallcurrent changes, with the integral of equation (4) providing for anaccurate time transformation.

[0055] The vertex finder 22 is configured, in this embodiment by the useof rotational matrices, to identify the vertices V of the equiphasepoints of the grouped sets of signal peaks SP₁, SP₂, SP₃ as determinedby the equiphase space-time map generator 20, where the components ofeach injected sample plug have a common vertex V by virtue of being timeand/or spatially separated in the space-time dimension. All of thesample components injected in a single sample plug are uniquelyidentified by a single vertex V in space-time co-ordinates, thusallowing for the identification of the sample components from each of aplurality of separately-provided sample plugs. FIG. 5 illustrates thevertex V as determined from the generated space-time map M. Thisspace-time map includes only a single vertex V as all of the componentswere provided in a single sample plug.

[0056] By using each vertex V as a constraint to extract the velocityspectrum of the sample components, the resolution is approximatelyproportional to {square root}n, where n is the number of components. Inthis way, the velocity of one component is calculated using thevelocities of all of the other components from the same sample plug, andthus, as the number of components in a sample increases, the resolutionof the analysis increases accordingly. Such space parameterisation whichresults in multiple vertex formation in the form of intensity enhancedregions in space-time co-ordinates is particularly suited to the casesof multiple sample injections and multiple column correlation. The powerof this technique has been demonstrated on DNA samples which includelarge numbers of fragments (>100) having lengths of one base pairdifference, thereby providing a sequencing technique having a greatlyextended dynamic range.

[0057] From the determination of the vertices V in the space-time map M,high resolution of the electrophoresis data is achieved, allowingaccurate determination of the velocities of the sample components asillustrated in FIG. 6.

[0058] Use of the above-described electrophoresis apparatus to sequenceDNA samples having the base pair terminations A, T, G and C will now bedescribed hereinbelow.

[0059] In use, four sample plugs comprising DNA bands having differentlength and one of base pair terminations A, T, G and C are separatelyintroduced into the ports 6, 8, 10, 12 of the separation channel 4, andelectrophoretically driven therealong. In one mode of use, the sampleplugs are introduced simultaneously into the ports 6, 8, 10, 12 whichare spatially separated along the separation channel 4. In another modeof use, the sample plugs are introduced sequentially into one of theports 6, 8, 10, 12 so as to be time spaced. The signals S₁, S₂, S₃, . .. , S_(n) detected by the detector 16 as the DNA bands pass thedetecting elements at the detecting positions z₁, z₂, Z₃, . . . , Z_(n)are collected by the data collector 18. The velocity sorter 19 thendetermines the nominal velocities v₁, v₂, v₃, . . . , V_(n) of thesample components associated with each of the signal peaks SP₁, SP₂,SP₃, . . . , SP_(n) of the signals S₁, S₂, S₃, . . . , S_(n) and groupsthose signal peaks SP₁, SP₂, SP₃, . . . , SP_(n) into sets according tonominal velocity. The equiphase space-time map generator 20 thendetermines the local minima of the signal peaks SP₁, SP₂, SP₃, . . . ,SP_(n) of the signals S₁, S₂, S₃, . . . , S_(n), and generates anequiphase space-time map M. The vertex finder 22 then identifies thevertices V_(A), V_(T), V_(G), V_(C) of the determined local minima foreach of the grouped sets of signal peaks SP₁, SP₂, SP₃, . . . , SP_(n).In this embodiment the space-time map M includes four vertices V_(A),V_(T), V_(G), V_(C) as four sample plugs were separately injected intothe separation channel 4, each being attributable to DNA bands havingone of the base pair terminations A, T, G and C. In this way, the DNAsample can be sequenced, with the lengths of the DNA bands beingdetermined from the migration velocities.

[0060] Finally, it will be understood that the present invention hasbeen described in its preferred embodiment and can be modified in manydifferent ways without departing from the scope of the invention asdefined by the appended claims.

1. A system for characterising, in particular sequencing, polymericsamples, comprising: a space-time map generator for generating aspace-time map of points representative of the signal peaks of signalsdetected at a plurality of spaced positions as passed by the migratingcomponents of a plurality of separately-provided sample plugs; and avertex finder for identifying vertices from the space-time map, with asingle vertex being identified for the components of eachseparately-provided sample plug and the velocities of the samplecomponents being determinable from the points in the space-time map. 2.The system of claim 1, further comprising a velocity sorter fordetermining the nominal velocities associated with the signal peaks inthe signals and grouping those signal peaks into sets according tonominal velocity.
 3. The system of claim 1 or 2, wherein the space-timemap generator is configured to utilise a corrected time component ingenerating the space-time map according to a function of the electriccurrent variation.
 4. The system of claim 3, wherein the correction isaccording to the function t_(c)=∫I₀/I(t′)dt′ in the range of 0 to t,where t is the measured time, t_(c) is the corrected time, I is themeasured current and I₀ is the reference current.
 5. The system of anyof claims 1 to 4, wherein the space-time map generator is an equiphasespace-time map generator for generating an equiphase space-time map ofequiphase points.
 6. The system of claim 5, wherein the equiphasespace-time map generator is configured to transform each data set into aset of local slopes and determine the local minima as the minimumabsolute local derivatives.
 7. The system of any of claims 1 to 6,wherein the components are non-labelled.
 8. The system of any of claims1 to 6, wherein the components are labelled.
 9. The system of any ofclaims 1 to 8, wherein the components are migrated through a channel.10. The system of claim 9, wherein the channel comprises a separationchannel through which the components are electrophoretically driven. 11.The system of any of claims 1 to 10, wherein the sample plugsrespectively comprise DNA bands having one of the base pairterminations.
 12. A method of characterising, in particular sequencing,polymeric samples, comprising the steps of: generating a space-time mapof points representative of the signal peaks of signals detected at aplurality of spaced positions as passed by the migrating components of aplurality of separately-provided sample plugs; and identifying verticesfrom the space-time map, with a single vertex being identified for thecomponents of each separately-provided sample plug and the velocities ofthe sample components being determinable from the points in thespace-time map.
 13. The method of claim 12, further comprising the stepsof determining the nominal velocities associated with the signal peaksin the signals and grouping those signal peaks into sets according tonominal velocity.
 14. The method of claim 12 or 13, wherein a timecomponent corrected according to a function of the electric currentvariation is utilised in generating the space-time map.
 15. The methodof claim 14, wherein the correction is according to the functiont_(c)=∫I₀/I(t′)dt′ in the range of 0 to t, where t is the measured time,t_(c) is the corrected time, I is the measured current and I₀ is thereference current.
 16. The method of any of claims 12 to 15, wherein thespace-time map is an equiphase space-time map of equiphase points. 17.The method of claim 16, wherein the equiphase points are determined bytransforming each data set into a set of local slopes and determiningthe local minima as the minimum absolute local derivatives.
 18. Themethod of any of claims 12 to 17, wherein the components arenon-labelled.
 19. The method of any of claims 12 to 17, wherein thecomponents are labelled.
 20. The method of any of claims 12 to 19,wherein the components are migrated through a channel.
 21. The method ofclaim 20, wherein the channel comprises a separation channel throughwhich the components are electrophoretically driven.
 22. The method ofany of claims 12 to 21, wherein the sample plugs are provided at spacedtime intervals.
 23. The method of any of claims 12 to 21, wherein thesample plugs are provided at spaced positions.
 24. The method of claim23, wherein the sample plugs are provided substantially at the sametime.
 25. The method of any of claims 12 to 24, wherein the sample plugsrespectively comprise DNA bands having one of the base pairterminations.
 26. An electrophoresis apparatus including the system ofany of claims 1 to
 11. 27. A system for characterising, in particularsequencing, polymeric samples substantially as hereinbefore describedwith reference to the accompanying drawings.
 28. A method ofcharacterising, in particular sequencing, polymeric samplessubstantially as hereinbefore described with reference to theaccompanying drawings.